The overall objective of the proposed research is to elucidate the mechanism of protein biosynthesis in cell organelles such as mitochondria and chloroplasts and to compare this system to prokaryotic and eukaryotic cytoplasmic protein synthesizing systems. Our first objective is to carry out a thorough investigation of the chloroplast protein synthesis elongation factors. We have obtained the Euglena gracilis protein synthesis translocase(EF-Gchl) in highly purified form and plan to carry out detailed studies of the similarities and differences between this factor and the comparable factor from the cytoplasm and mitochondria of the same cell. In addition, we will seek to determine how this cytoplasmically synthesized organellar protein is transported into the chloroplast. We have also partially purified the chloroplast translational factors responsible for the binding of aminoacyl-tRNA to ribosomes during the elongation steps of protein synthesis. We now seek to purify and thoroughly characterize these factors and to compare them to prokaryotic and cytoplasmic elongation factors. Our second objective is to establish an in vitro system from the chloroplasts of Euglena gracilis which will translate natural messenger RNAs. We then propose to purify the initiation factors and to define their roles in the initiation process. These studies will permit us to compare the process of chain initiation in cell organelles, bacteria and the cytoplasm of eukaryotic cells. A long-term objective is to develop a defined in vitro system in which to study the regulation of organelle protein biosynthesis and its integration into the complex metabolism of the eukaryotic cell.